Proliferation, Characterization and Differentiation Potency of Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) Cultured in Fresh Frozen and non-Fresh Frozen Plasma

Mesenchymal stem cells (MSCs) have unique properties, including high proliferation rates, self-renewal, and multilineage differentiation ability. Their characteristics are affected by increasing age and microenvironment. This research is aimed to determine the proliferation, characteristics and differentiation capacity of adipose tissue-derived (AT)-MSCs at many passages with different media. The cell proliferation capacity was assayed using trypan blue. MSCs characterization (CD90, CD44, CD105, CD73, CD11b, CD19, CD34, CD45, and HLA-DR) was performed by flow cytometry, and cell differentiation was determined by specific stainings. Population doubling time (PDT) of AT-MSCs treated with fresh frozen plasma (FFP) and non-FFP increased in the late passage (P) (P15 FFP was 22.67 ± 7.01 days and non-FFP was 19.65 ± 2.27 days). Cumulative cell number was significantly different between FFP and non-FFP at P5, 10, 15. AT-MSCs at P4-15 were positive for CD90, CD44, CD105, and CD73, and negative for CD11b, CD19, CD34, CD45, and HLA-DR surface markers. AT-MSCs at P5, 10, 15 had potential toward adipogenic, chondrogenic, and osteogenic differentiation. Therefore, PDT was affected by increased age but no difference was observed in morphology, surface markers and differentiation capacity among passages. Cumulative cell number in FFP was higher in comparison with non-FFP in P5, 10, 15. Our data suggest that FFP may replace FBS for culturing MSCs.

The waste of liposuction surgery is the best source of MSCs because AT from this waste contains abundant AT-MSCs, with low side effects for the donor. AT-MSCs has a fibroblast-like morphology, and has the potential to differentiate into chondrocyte, adipocyte, and osteocyte (12).
AT had been isolated and characterized since 1970. It had been obtained from bovine, dog, goat, horse, rabbit, rat, mouse, pig, and human.
Difference in liposuction procedures, isolation techniques, culture conditions, age, and body mass index impact the AT-MSCs isolation yield.

Passaging and cell proliferation analysis
Passaging MSCs with FFP treatment were cultured 3-5 days in FFP medium, with the medium being replaced every 2 days. For non-FFP, cells were given a complete medium consisting of 80% MEM-α, 1% antibiotic and antimicotic, and 1% heparin but at the last day the cells were treated with a medium without FFP for 24 h to make the cells starve. where NI is the inoculum cell number and NH is the cell harvest number. Then, to determine cumulative PD data, the PD at the previous passage was added.
The PD time (PDT) was determined by the formula(4).

Characterization of MSCs
MSCs from P3-P14 cultured at density of 2 x

Statistical analysis
Statistical analysis was conducted using SPSS software (version 20.0). Data were presented as mean ± standard deviation. Significant differences among treatments were determined using the oneway analysis of variance (ANOVA) and P <0.05 was considered as statistically significant, along with Tukey post hoc test. The PDT and PD rates are represented in Cell morphology is illustrated in Figure 2 Figure 3). FFP supplement proved that it was effective to increase proliferation and did not affect the characteristics of MSCs.    Figure   4B). Chondrogenic differentiation was visualized by alcian blue staining ( Figure 4C).

Discussion
In a previous study, PD of umbilical cor blood they were stopped at P11-12 (19). In comparison with our results, the proliferation of AT-MSCs were stable and high at P1 to 8 for FFP and P1 to 9 for non-FFP medium, then it decreased at P 10 to 15.
In the present study, the cell proliferation and cell number were affected by passage and microenvironment. A significant difference was observed between FFP and non-FFP in P4, P5 for both PDT and PD rate (Table 1) 10% FBS-supplemented culture (24). Human blood-derived components such as human serum (autologous or pooled allogeneic), platelet products, and umbilical cord blood serum seem to be the most efficient and safer, and can be considered a promising FBS alternative (25,26). In this study, FFP was used as supplement medium. FBS in MSCs may pose an infection risk, which induce immunological reactions in the host for therapy application (27). Thus, regarding a significant safety concern when administering MSCs cultured in FBS, many researchers try to change the FBS use in MSCs culture for therapy application. It has also been reported that patients who have received cell transplantation with MSCs expanded in FBS exhibited antibodies against bovine antigens (28).
The present data showed that the proliferation of AT-MSCs was higher using FFP than non-FFP.
At P4, the cumulative cell number for FFP treatment was higher than non-FFP treament ( Table   1). The human AB serum and tPRP are alternatives to FCS for AT-MSCs (24). In another study, autologous serum increased the proliferation rates (29). PRP can produce chemokines; cytokines and growth factors can promote the recruitment, adhesion, and proliferation of adult stem cells (30).
To eliminate the use of animal products in human AT-derived stem cells (ADSCs) cultures, PRP can substitute the FBS as PRP contains a wide range of proteins, growth factors, and enzymes supporting attachment, growth, and proliferation of cells (31,32).
Based on data presented in Figure 1